Zoonotic Abbreviata Caucasica in Wild Chimpanzees (Pan Troglodytes Verus) from Senegal

pathogens

Article Zoonotic Abbreviata caucasica in Wild Chimpanzees (Pan troglodytes verus) from Senegal

Younes Laidoudi 1,2 , Hacène Medkour 1,2 , Maria Stefania Latrofa 3, Bernard Davoust 1,2, Georges Diatta 2,4,5, Cheikh Sokhna 2,4,5, Amanda Barciela 6 , R. Adriana Hernandez-Aguilar 6,7 , Didier Raoult 1,2, Domenico Otranto 3 and Oleg Mediannikov 1,2,*

1 IRD, AP-HM, Microbes, Evolution, Phylogeny and Infection (MEPHI), IHU Méditerranée Infection, Aix Marseille Univ, 19-21, Bd Jean Moulin, 13005 Marseille, France; [email protected] (Y.L.); [email protected] (H.M.); [email protected] (B.D.); [email protected] (D.R.) 2 IHU Méditerranée Infection, 19-21, Bd Jean Moulin, 13005 Marseille, France; [email protected] (G.D.); [email protected] (C.S.) 3 Department of Veterinary Medicine, University of Bari, 70010 Valenzano, Italy; [email protected] (M.S.L.); [email protected] (D.O.) 4 IRD, SSA, APHM, VITROME, IHU Méditerranée Infection, Aix-Marseille University, 19-21, Bd Jean Moulin, 13005 Marseille, France 5 VITROME, IRD 257, Campus International UCAD-IRD, Hann, Dakar, Senegal 6 Jane Goodall Institute Spain and Senegal, Dindefelo Biological Station, Dindefelo, Kedougou, Senegal; [email protected] (A.B.); [email protected] (R.A.H.-A.) 7 Department of Social Psychology and Quantitative Psychology, Faculty of Psychology, University of Barcelona, Passeig de la Vall d’Hebron 171, 08035 Barcelona, Spain * Correspondence: [email protected]; Tel.: +33-041-373-2401

Received: 19 April 2020; Accepted: 23 June 2020; Published: 27 June 2020

Abstract: Abbreviata caucasica (syn. Physaloptera mordens) has been reported in human and various non-human primates including great apes. The identification of this nematode is seldom performed and relies on egg characterization at the coproscopy, in the absence of any molecular tool. Following the recovery of two adult females of A. caucasica from the feces of wild Senegalese chimpanzees, morphometric characteristics were reported and new data on the width of the esophagus (0.268–0.287 mm) and on the cuticle structure (0.70–0.122 mm) were provided. The molecular characterization of a set of mitochondrial (cox1, 16S rRNA, 12S rRNA) and nuclear (18S rRNA and ITS2) partial genes was performed. Our phylogenetic analysis indicates for the first time that A. caucasica is monophyletic with Physaloptera species. A novel molecular tool was developed for the routine diagnosis of A. caucasica and the surveillance of Nematoda infestations. An A. caucasica-specific qPCR targeting the 12S gene was assessed. The assay was able to detect up to 1.13 10 3 eggs/g × − of fecal matter irrespective of its consistency, with an efficiency of 101.8% and a perfect adjustment (R2 = 0.99). The infection rate by A. caucasica in the chimpanzee fecal samples was 52.08%. Only 6.19% of the environmental samples were positive for nematode DNA and any for A. caucasica. Our findings indicate the need for further studies to clarify the epidemiology, circulation, life cycle, and possible pathological effects of this infestation using the molecular tool herein developed.

Keywords: Abbreviata caucasica; Physaloptera mordens; Pan troglodytes verus; wild chimpanzees; nematode; zoonosis; Senegal

1. Introduction Physalopteriasis is caused by parasitic nematodes from the genus Physaloptera (Spirurida, Physalopteridae) [1], which has been distributed in Africa and the Middle East (i.e., Iran) since

Pathogens 2020, 9, 517; doi:10.3390/pathogens9070517 www.mdpi.com/journal/pathogens Pathogens 2020, 9, 517 2 of 22 prehistoric times [2,3]. Following the first formal description of the Physaloptera genus (Rudolphi in 1819), Physaloptera abbreviata (Rudolphi, 1819) was designated as the type species [4]. Afterward, Abbreviata was defined as a distinct genus, based on the number, mode, and origin of the uteri, which constitute the main keys for genus differentiation [4]. Adult stages of this genus are found in the stomach of a variety of animals such as reptiles and mammals including humans [5]. Abbreviata (=Physaloptera) caucasica (Linstow 1902), is a gastrointestinal nematode of Simiiformes (Anthropoidea) members [6]. After its discovery in a Caucasian man, Linstow (1902) provided an incomplete description with some erroneous morphological details [7]. In 1926, Schulz gave the complete description of A. caucasica after re-examining the original specimens, establishing a close relationship of this species with Physaloptera mordens (Leiper, 1908) isolated from humans in Central Africa. The unique difference identified among specimens was the presence/absence of a series of small teeth between and exterior to the large teeth on the inner face of the lip of A. caucasica and P. mordens, respectively [8]. A few months later, Ortlepp re-examined P. mordens and confirmed that the small teeth were missing in the previous examinations [9], therefore synonymizing P. mordens (Leiper, 1908) and A. caucasica (Linstow, 1902). The A. caucasica infection has been reported in New and Old World monkeys [10], rhesus macaques (Macaca mulatta), baboons (Papio spp.), and great apes including both captive (Pongo spp.) and wild (Pongo abelii)[1,11]. It has also been reported in chimpanzees (Pan troglodytes) from Gombe (Tanzania) and Ngogo (Uganda) [12,13], although no adult specimens were examined. Furthermore, eggs of Physaloptera sp. have been reported in wild Senegalese chimpanzees [12,14]. Human and non-human primates probably constitute the natural host for A. caucasica [8]. The adult parasite looks like Ascaris sp. under the naked eye [15] and occurs in the digestive tract from the esophagus to the small intestine, where it can induce serious disease manifestations such as abdominal pain, anorexia, vomiting, and bloody diarrhea [2]. Records of the clinical signs in infected chimpanzees are lacking [6]. Nowadays, the detection of A. caucasica depends on the identification of eggs in the feces or in the detection of adult stages during post-mortem examination of the gastrointestinal tract of infested hosts [1]. The arthropod intermediate and/or paratenic hosts remain unknown though some experimental evidence indicates that A. caucasica could develop to the infective stage in Blatella germanica and in Schistocerca gregaria [5]. For other Physaloptera species, the intermediate arthropod host has been assessed such as Tribolium confusum [16], ground beetles, Harpalus sp. [17] as well as crickets, Acheta assimilis, and grasshoppers, Melanoplus femurrubrum [18,19]. Under natural conditions, A. caucasica may develop in arthropods and infestation probably occurs through ingestion of beetles, crickets, or other arthropods as well as paratenic hosts containing infective larvae [8,15]. However, the potential involvement of up to 28 paratenic and second intermediate hosts is suspected [6]. Anthelminthic drugs have been reported to be effective for the treatment of physalopteriasis in non-human primates [1]. However, their control should be reinforced by a molecular characterization to avoid the misleading conclusions about this parasite, sanitation and control of the potential paratenic or arthropod hosts as well as the surveillance of the infestation from the colon of non-human primates [1,16]. As part of the control of infectious and zoonotic diseases in the current chimpanzee population from the Dindefelo Community Natural Reserve in Senegal, we present here morphometric and phylogenetic findings to support the occurrence of A. caucasica in chimpanzees, providing a molecular characterization of a set of target mitochondrial (cox1, 16S rRNA, 12S rRNA) and nuclear (18S rRNA and ITS2) genes and morphological identification of adult specimens collected from feces of West African chimpanzees in Senegal. In addition, we developed a molecular test that could be used in a routine diagnostic laboratory instead of the labor-intensive coprological methods. The molecular test provides detection, egg quantification, and genetic characterization of A. caucasica from biological samples. We therefore applied this tool to a surveillance process and molecular xenomonitoring of A. caucasica from possible intermediate hosts and our current population of West African chimpanzees from Senegal. Pathogens 2020, 9, 517 3 of 22

2. Results

2.1. Morphological Characteristics of Adult A. caucasica Comparative measurements of A. caucasica adult females from our study with A. caucasica (Linstow, 1902) and its synonymous species P. mordens (Lipper, 1908) [8] are detailed in Table1. Two female complete specimens measuring 54.7 mm and 59.6 mm in length and 2.08 mm and 2.13 mm in width, respectively, were examined. The nematodes were characterized by the anterior end with a short buccal cavity (Figure1(1a)) and by a cuticle reﬂecting over the lips to form a cephalic collarette (Figure1(1b)) with two lateral pseudolabia undivided (Figure1(1c,1d)). Nerve ring at 0.430 mm from the anterior end. The esophagus consisting of two parts: muscular esophagus 0.75 mm long (Figure1(2a)) and 0.287 mm wide, 0.79 mm long, and 0.268 mm wide, respectively. The esophagus total length was 5.52 mm and 4.82 mm, respectively in two samples. In the mid-body, the cuticle was 0.70–0.122 mm thick and ﬁnely striated (Figure1(2b)). The worms showed the presence of two small symmetrical pins in the front third of the body (Figure1(2c)). Vulva 1.560 mm from anterior end (Figure1(2d)). Presence of four uteri (Figure1(3a)). Eggs were small 36–41 µm 28–32 µm (Figure1(3b)). Tail length was 1.084 mm (Figure1(3c)). The caudal end showed the × presence of a caudal appendix (Figure1(3d)). Pathogens 2020, 9, 517 4 of 22

Table 1. Comparative measurement (in mm unless speciﬁed) of adult female of A. caucasica from our study with A. caucasica (Linstow, 1902) and its synonymous species P. mordens (Lipper, 1908) according to Fain and Vandepitte (1964) [7], Linstow (1902) and Schulz (1926) [8].

Measurement of Fain and Measurement of This Study Measurement of Schulz (1926) Vandepitte (1964) Linstow, 1902 A. caucasica (Syn. P. A. caucasica: Measures A. caucasica A. caucasica: Type Species P. Mordens: Type Species mordens) Type Species Chimpanzee (Senegal) Men (Congo) Men (caucasia) Men (caucasia) Men (Uganda) Organ Segment CHS 11 CHS 31 N1 N1 Adult Female N1 N2 N1 N2 Length 54.7 59.6 108 117 27 24.75 23.84 41 100

Body Width 2.08 2.13 2 3 1.14 1.18 1.12 1.8 2.8 Index a 26.3 28 54 39 23.68 20.97 21.29 22.78 35.71 From the Nerve ring 0.43 - 0.7 0.78 - 0.454 0.454 - - anterior end Total length 5.52 4.82 9 11 - 3.5 3.72 - - Length Esophagus 0.79 0.75 0.6 0.6 - 0.43 0.35 - - muscolar (e) (e) Width 0.268 0.287 - - - - - Index b 9.91 12.37 12 10.63 - 7 6.4 6.2 6.2 Cuticle Width 0.92–0.102 0.70–0.122 ------From the Vulva 1.56 - 21 23 - 3.50 4.62 - - anterior end Eggs (µm) 37–41 28–32 36–39 28–31 60–65 45–55 57 39 57–62 42–45 45–49 32–34 × × × × × × Tail 1.084 - 1.3 1.4 0.51 0.578 0.532 - - Tail Index c 50.46 - 80 83 53 43 45 70 90 CHS 11 and CHS31: Code sample of specimens from the present study. N1, N2: Code samples of specimens from the study of Schulz (1926). Index a, b, and c are the ratio of body length to body width, esophagus length and tail length, respectively. Pathogens 2020, 9, 517 5 of 22

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Figure 1. Cont.

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FigureFigure 1. 1. LightLight microscopymicroscopy images of of A.A. caucasica caucasica adultadult females. females. (1a (1a) Buccal) Buccal cavity. cavity. (1b ()1b Cephalic) Cephalic Figurecollarette.collarette. 1. Light ((1c1c,, 1d 1dmicroscopy)) TheThe twotwo laterallateralimages pseudolabia. of A. caucasica (2a (2a) adult)Esophagus. Esophagus. females. (2b ( 2b)( 1aThick)) Thick Buccal finely ﬁnely cavity. striated striated (1b from) Cephalic from the the collarette.mid-body.mid-body. ( (1c 2c(2c,) )1d Pins. Pins.) The ((2d two) Vulva. lateral ( (3a3a pseudolabia.)) Uteri. Uteri. (3b (3b) )Eggs. Eggs.(2a) (3cEsophagus. (3c) Tail.) Tail. (3d (3d) (Caudal2b) Caudal) Thick appendix. appendix. finely striated from the mid-body. (2c) Pins. (2d) Vulva. (3a) Uteri. (3b) Eggs. (3c) Tail. (3d) Caudal appendix. 2.2.2.2. A. A. caucasica caucasica EggsEggs fromfrom Positive Feces Feces 2.2. A.Eggs Eggscaucasica of ofA. A. caucasicaEggscaucasica from were Positive identified identiﬁed Feces morphologically morphologically from from two two fecal fecal samples samples taken taken from from animals animals foundfoundEggs infested infested of A. withcaucasica with adult adult were worms. worms. identified The The eggs morphologicallyeggs were were apparently apparently from identical twoidentical fecal to the samplesto micrographthe micrograph taken offromA. ofcaucasicaanimals A. foundeggscaucasica reported infested eggs elsewhere reportedwith adult elsewhere [7]. worms. Eggs were[7]. The Eggs embryonated eggs were were em bryonatedapparently and had aand characteristicidentical had a characteristic to thickthe micrograph shell thick and shell hyaline of A. caucasicacoatand (Figure hyaline eggs2 ).coat reported (Figure elsewhere 2). [7]. Eggs were embryonated and had a characteristic thick shell and hyaline coat (Figure 2).

Figure 2. Coproscopy showing the A. caucasica eggs found in wild chimpanzee feces (formol-ether method, 100× magnification). Figure 2. Coproscopy showing showing the the A. caucasica eggs found in wild chimpanzee feces (formol-ether 2.3.method, Molecular 100× 100 Characterization magnification).magniﬁcation). of Adult A. caucasica Worms × First, nearly full-length DNA sequences of the 18S rRNA (AN: MN956824, MN956825), ITS2 2.3.(AN: Molecular MN956809, Characterization MN956810), of Adultcox1 A.(AN: caucasica MT231294, Worms MT231295), 16S rRNA (AN: MN956826, First, nearly full-length DNA sequences of the 18S rRNA (AN: MN956824, MN956825), ITS2 (AN: MN956809, MN956810), cox1 (AN: MT231294, MT231295), 16S rRNA (AN: MN956826,

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2.3. Molecular Characterization of Adult A. caucasica Worms First, nearly full-length DNA sequences of the 18S rRNA (AN: MN956824, MN956825), ITS2 (AN: MN956809, MN956810), cox1 (AN: MT231294, MT231295), 16S rRNA (AN: MN956826, MN956827), and 12S rRNA (AN: MN956811, MN956812) genes were obtained from adult worms of A. caucasica. The sequences of each gene were identical to each other. The BLAST analysis of 1140 bp of the 18S rRNA gene showed the highest query cover (100%) with eight sequences of Physaloptera sp. A nucleotide identity of 97.9% (1118/1142) was observed with Physaloptera apivori (EU004817) isolated from birds in Germany, followed by 97.89% (1116/1140) for both Physaloptera sp. (HM067978) isolated from long-tailed macaques (Macaca fascicularis) in China, and Physaloptera turgida (DQ503459) isolated from North American opossums (Didelphis virginiana) in Louisiana, USA. Finally, an identity ranging from 97.46% to 97.81% was observed within the five other sequences of Physaloptera species isolated from reptiles and mammals (EF180069, MG808040, JF934734, AY702703, and EF180065). In contrast, the BLAST analysis of the partial (759 bp) cox1 nucleotide sequence showed the lowest values of query cover and identity with those of Physaloptera spp. from the GenBank database, having a greater sequence coverage (i.e., 98 to 100%) with those of Onchocercidae members than that observed for Physaloptera species (i.e., 83 to 88%). Among Physaloptera, the highest nucleotide identity values observed were 83.7% (529/632) with Physaloptera sp. (MH752202) isolated from brown anoles (Anolis sagrei) in the USA, 83.5% (530/635) with both P. turgida (KT894808) and Turgida sp. (KC130680) isolated from opossums (Didelphis spp.) in Brazil and Mexico, respectively, and 83.2% (558/671) with P. amazonica (MK309356) isolated from Gardner’s spiny rat (Proechimys gardneri) in Brazil, whilst lower identity values, ranging from 82.1% to 82.9%, were observed for the other five sequences of Physaloptera species (MH782844, KT894803, KT894804, KP981418, KT894805). In contrast, the Physaloptera cox1 amino acid sequence appeared first among the top ten sequences of BLASTx [20]. Abbreviata caucasica COI sequence (protein id: QIP66136) showed an identity of 88.1% with P. retusa (AMX28288) isolated from golden tegu (Tupinambis teguixin) in Brazil, 87.7% with P. mirandai (AMX28289) isolated from brown four-eye opossums (Metachirus nudicaudatus) in Brazil with a coverage of 86% for both, 87.5% of identity and 98% of coverage with P. rara (QDF64304) isolated from dogs (Canis lupus familiaris), and 87.2% of identity and 86% of coverage with Physaloptera sp. (AMX28292), P. bispiculata (AMX28291), P. amazonica (QDX15779), and P. hispida (QCF40948). BLASTn analyses of 16S rRNA (416 bp) and 12S rRNA (573 bp) sequences identified the first 60 sequences that corresponded to those of Filarioidea and Thelazidae without any Physaloptera. Nucleotide identity of about 75% with a query coverage of more than 99% were observed among these Spiriruds. Finally, the BLASTn analysis of the partial (675 bp) sequence of the ITS2 showed an identity of 93.37% (67/71) and a coverage of 10% with the unique GenBank sequence of Physaloptera alata (AY702694) isolated from birds. The interspecific nucleotide pairwise (INP) distance of the 18S rRNA, cox1, 16S rRNA, 12S rRNA, and ITS2 of A. caucasica within Physalopteridae members are shown in Table S1. All sequences were well resolved in the chromatograms. The partial cox1 sequence was correctly aligned against the complete cox1 sequence (MH931178) of P. rara and no stop codon was observed in the translated amino-acid sequences, suggesting the absence of co-amplified numts. Furthermore, sequence alignment of COI with those of Physaloptera species showed nineteen amino-acid changes specific for A. caucasica (Figure S1). The interspecific nucleotide pairwise (INP) distance among the 645 bp of cox1 corroborated with the IaaP distance, among the corresponding 208 amino acid (Figure3) and was substantially higher (ten times) between A. caucasica and Physalopteridae members in comparison with the 18S rRNA sequences. Pathogens 2020, 9, 517 8 of 22 Pathogens 2020, 9, x FOR PEER REVIEW 8 of 21

Figure 3. Scatter chart showing the interspeciﬁc pairwise distance between the COI sequence of Figure 3. Scatter chart showing the interspecific pairwise distance between the COI sequence of A. A. caucasica and other nematodes based on both IaaPD and INPD. The INP distance was 0.31 (Std Err: 0.03) caucasica and other nematodes based on both IaaPD and INPD. The INP distance was 0.31 (Std Err: and 0.210.03) (Std and Err: 0.21 0.06) (Std betweenErr: 0.06)A. between caucasica A.and caucasicaHeliconema and Heliconema longissimum longissimumfor THE 12S for rRNATHE 12S (GQ332423) rRNA and 16S(GQ332423) rRNA (GQ332423) and 16S rRNA sequences, (GQ332423) respectively. sequences, for resp the ITS2ectively. sequences, for the theITS2 INP sequences, distance the observed INP betweendistanceA. caucasicaobservedand between Filarioidea A. caucasica (XR 002251420, and Filarioidea JQ316671, (XR FM206482,002251420, JQ316671, DQ317666, FM206482, DQ317657, and DQ317652),DQ317666, DQ317657, Spirocercidae and (MH038181),DQ317652), Spirocercidae Habronematidae (MH038181), (MH038181), Habronematidae and Gongylonematidae (MH038181), (LC026032,and Gongylonematidae LC278392, and LC026029)(LC026032, membersLC278392, ranged and LC026 from029) 0.51 members (Std Err: ranged 0.06) to from 0.54 0.51 (Std (Std Err: Err: 0.06). No ITS20.06) sequences to 0.54 (Std of Err: Physalopteroidea 0.06). No ITS2 sequences superfamily of Physalopteroidea were available. superfamily were available.

The BayesianThe Bayesian trees trees inferred inferred from fromcox1, cox nucleotide,1, nucleotide, and and protein protein sequences, sequences, and and from from 18S rRNA18S rRNA genes are showngenes inare Figure shown4A,B in Figures and Figure 4A,B5, respectively.and 5, respectively. All phylograms All phylograms provide provide evidence evidence that A. that caucasica A. is ancaucasica integral is part an ofintegral the genus part Physalopteraof the genus. InPhysaloptera particular,. In on particular, the cox1 tree, on theA. caucasicacox1 tree,clustered A. caucasica with Physalopteraclusteredsp. with (MH752202) Physaloptera andsp. (MH752202)P. retusa (KT894803) and P. retusa isolated (KT894803) respectively isolated respectively from Anolis from sagrei Anolisin the USAsagrei and Tupinambisin the USA and teguixin Tupinambisin Brazil teguixin (Figure in Brazil4A). Similarly,(Figure 4A). on Similarly, the COI on tree, the COIA. caucasica tree, A. caucasicaclustered withclusteredP. rara (QDF64304), with P. rara (QDF64304),P. retusa (AMX28288), P. retusa (AMX28288),Physaloptera Physalopteraspp. (QEQ27063, spp. (QEQ27063, AYA23053), AYA23053),P. turgida P. turgida (AMX28293), and Turgida sp. (AFZ99495) (Figure 4B), while on the 18S rRNA tree, A. (AMX28293), and Turgida sp. (AFZ99495) (Figure4B), while on the 18S rRNA tree, A. caucasica clustered caucasica clustered together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) isolated from birds in isolated from birds in Germany (Figure 5). Germany (Figure5). In addition, all Physaloptera and A. caucasica haplotypes shared a Euler circuit in the Templeton– Crandall–Sing (TCS) network tree for cox1 sequences. Abbreviata caucasica was connected by three-step branches to the Euler circuit, while all Physaloptera haplotypes were connected to the circuit by one to three-step branches (Figure6). Hence, the TCS network analysis replicates the same results observed in the Bayesian inferences.

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Figure 3. Scatter chart showing the interspecific pairwise distance between the COI sequence of A. caucasica and other nematodes based on both IaaPD and INPD. The INP distance was 0.31 (Std Err: 0.03) and 0.21 (Std Err: 0.06) between A. caucasica and Heliconema longissimum for THE 12S rRNA (GQ332423) and 16S rRNA (GQ332423) sequences, respectively. for the ITS2 sequences, the INP distance observed between A. caucasica and Filarioidea (XR 002251420, JQ316671, FM206482, DQ317666, DQ317657, and DQ317652), Spirocercidae (MH038181), Habronematidae (MH038181), and Gongylonematidae (LC026032, LC278392, and LC026029) members ranged from 0.51 (Std Err: 0.06) to 0.54 (Std Err: 0.06). No ITS2 sequences of Physalopteroidea superfamily were available.

The Bayesian trees inferred from cox1, nucleotide, and protein sequences, and from 18S rRNA genes are shown in Figures 4A,B and 5, respectively. All phylograms provide evidence that A. caucasica is an integral part of the genus Physaloptera. In particular, on the cox1 tree, A. caucasica clustered with Physaloptera sp. (MH752202) and P. retusa (KT894803) isolated respectively from Anolis sagrei in the USA and Tupinambis teguixin in Brazil (Figure 4A). Similarly, on the COI tree, A. caucasica clustered with P. rara (QDF64304), P. retusa (AMX28288), Physaloptera spp. (QEQ27063, AYA23053), P. turgida (AMX28293), and Turgida sp. (AFZ99495) (Figure 4B), while on the 18S rRNA tree, A. Pathogenscaucasica2020 clustered, 9, 517 together with Physaloptera apivori (EU004817) and Physaloptera alata (AY702703) 9 of 22 isolated from birds in Germany (Figure 5).

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Figure 4. Phylogram generated from Bayesian inference. (A) Based on 651 bp from nucleotide sequences of the cox1 gene. (B) Based on 210 amino-acid from the COI-protein sequences. Numbers aboveFigure and 4. Phylogram below branches generated are fromthe display Bayesian of inference. nod statistics (A) Based and branch on 651 bp leng fromth, nucleotiderespectively. sequences Host, geographicalof the cox1 gene. location (B) Based (when on 210available), amino-acid and fromGenBank the COI-protein accession sequences.numbers and Numbers protein-id above are and indicated.below branches The identity are the ofdisplay each taxa of is nod colo statisticsr-coded andaccording branch to length, the genus. respectively. Likelihood Host, was geographical−5448.2 and −location1802.86 for (when nucleotide available), and and protein GenBank inferences, accession respectively. numbers and protein-id are indicated. The identity of each taxa is color-coded according to the genus. Likelihood was 5448.2 and 1802.86 for nucleotide − − and protein inferences, respectively.

Figure 5. Phylogram generated from Bayesian inference, based on 1209 bp from 18S rRNA sequences. Numbers above and below branches are the display of nod statistics and branches length, respectively. FigureHost, geographical5. Phylogram location generated (when from available), Bayesian and inference, GenBank based accession on 1209 numbers bp from are 18S indicated. rRNA sequences. Likelihood Numberswas 3466.3. above and below branches are the display of nod statistics and branches length, − respectively. Host, geographical location (when available), and GenBank accession numbers are indicated. Likelihood was −3466.3.

In addition, all Physaloptera and A. caucasica haplotypes shared a Euler circuit in the Templeton– Crandall–Sing (TCS) network tree for cox1 sequences. Abbreviata caucasica was connected by three- step branches to the Euler circuit, while all Physaloptera haplotypes were connected to the circuit by one to three-step branches (Figure 6). Hence, the TCS network analysis replicates the same results observed in the Bayesian inferences.

Pathogens 2020, 9, 517 10 of 22 Pathogens 2020, 9, x FOR PEER REVIEW 10 of 21

FigureFigure 6.6. Templeton–Crandall–Sing (TCS (TCS)) spanning network of the cox1 gene (651 bp) fragment.fragment. ColoredColored andand greyishgreyish circlescircles correspondcorrespond toto aa speciesspecies genotypegenotype or hypothetical genotype, respectively. ModelModel characteristicscharacteristics were: were: nucleotide nucleotide diversity diversity (pi =(pi1.282), = 1.282), number number of segregating of segregating sites (361), sites number (361), ofnumber parsimony-informative of parsimony-informative sites (242), sites Tajima’s (242), DTajima’s statistic D (D statistic= 30.3099), (D = 30.3099), and p (“D and>= p30.3099” (“D >= 30.3099”= 0). 2.4. Molecular= 0). Investigation of A. caucasica and Nematode Infestation from Biological Samples

2.3. MolecularThe molecular Investigation tool developed of A. caucasica in the and present Nematode study Infest wasation specific from for Biological the target Samples DNA without any amplification from the negative controls. The molecular tool developed in the present study was specific for the target DNA without any Results of the molecular screening for A. caucasica and nematode DNA are detailed in Table2. Among amplification from the negative controls. the 48 fecal samples tested, 52.08% (n = 25) were positive for A. caucasica, while all environmental samples Results of the molecular screening for A. caucasica and nematode DNA are detailed in Table 2. tested negative. In addition to the samples that tested positive for A. caucasica, the pan-Nematoda qPCR Among the 48 fecal samples tested, 52.08% (n = 25) were positive for A. caucasica, while all assay allowed for the detection of 29.17% (n = 14) of other fecal samples and 6.2% (n = 7, three soil samples environmental samples tested negative. In addition to the samples that tested positive for A. caucasica, from termite mounds and four termite specimens) of positive environmental samples. the pan-Nematoda qPCR assay allowed for the detection of 29.17% (n = 14) of other fecal samples and 6.2% (nTable = 7, 2.threeAbbreviata soil caucasicasamplesand from nematode termite infestations mounds regardingand four origin termite and fecalspecimens) consistency. of positive environmental samples. Infestation Rate (%) by qPCRs Fisher’s exact test showed that thereTested were Samplesno significant effects of localities and fecal consistency on nematodes and A. caucasica prevalences (Table 2). A. caucasica Nematodes Localities Table 2. AbbreviataLocality caucasica 1 and nematode 3infestations regarding 0 origin and fecal 33.3 consistency. Locality 2 6 66.7 100 Locality 3 39Infestation 53.8 Rate (%) by 82.1 qPCRs Tested Samples Total 48A. caucasica 56.3 Nematodes 81.3 Comparison by localities Fisher test (p) 0.148 0.052 Localities Fecal consistency LocalityFresh 1 383 40.00 33.3 70.0 LocalityDegraded 2 106 66.7 55.3 84.2100 FisherLocality test (p)3 39// 53.80.49 82.1 0.30 Total 48 56.3 81.3 Fisher’sComparison exact test showed by localities that there Fisher were no test significant (p) effects0.148 of localities and0.052 fecal consistency on nematodes andFecalA. consistency caucasica prevalences (Table2). Abbreviata caucasicaFresh cox1 species-specific primers38 successfully40.0 amplified a partial70.0 sequence (504 bp) from 84% (21/25) ofDegraded samples identified as positive10 by the qPCR targeting55.3 the 12S rRNA84.2 gene. There was no significant diffFishererence betweentest (p) both assays according// to the McNemar0.49 test (p = 0.25).0.30 All sequences

Abbreviata caucasica cox1 species-specific primers successfully amplified a partial sequence (504 bp) from 84% (21/25) of samples identified as positive by the qPCR targeting the 12S rRNA gene. There was no significant difference between both assays according to the McNemar test (p = 0.25). All

Pathogens 2020, 9, 517 11 of 22 were identical to each other and showed 100% similarity to those from adult specimens ampliﬁed with pan-Nematoda primers. All sequences were deposited in the GenBank database under the following accession numbers: MT231296–MT231316.

2.5. The Analytical Sensitivity of A. caucasica 12S rRNA qPCR and Egg Counting The performance characteristics of the 12S rRNA qPCR are shown in Table S2 and Figure S2. The assay was species specific and was able to detect up to 1.13 10 3 eggs/g of positive fecal samples × − (i.e., corresponding to 1.13 10 5 eggs/5 µL of DNA). The qPCR efficiency was 101.8% with 3.28 and × − − 28.68 as a Slope and Y-intercept values, respectively, allowing a perfect adjustment (R2 = 0.99). Table3 compares the A. caucasica eggs quantified by qPCR in terms of fecal consistency (fresh or degraded samples). Egg concentration in degraded feces (n = 4) was low (<1/g), but was higher (mean = 1.4 egg/g) in fresh feces (n = 21), while no effect of fecal consistency on egg concentration was observed (ANOVA, R2 = 0.032, Pr > F = 0.403).

Table 3. Abbreviata caucasica egg output from the positive samples.

Number of Positive Quantiﬁcation (Means Eggs/g) from Positive Fecal Consistency Samples Samples Degraded 4 0.2 Fresh 21 * 1.4 R2 0.032 Statistics One-way ANOVA Pr > F 0.403 *: one sample with an abnormal residual was removed before statistical analysis. Degraded: decomposing fecal samples.

3. Discussion In this study, we report on the presence of A. caucasica (adults and eggs) in the feces of western chimpanzees from Senegal. Our data indicate that this population of chimpanzees is exposed to a high nematode infestation (81.3%), particularly A. caucasica (52.1%). This corroborates previous data from chimpanzees in southeastern Senegal, in which the reported nematode species-specific prevalence was between 0.78% to 31% where Physaloptera sp. was often the most prevalent species (13.26 to 31%) [12,14]. However, it was not specified whether these Physaloptera sp. were A. caucasica or author Physaloptera species. Perhaps the use of molecular assays, which were not applied in these studies, could offer a better species resolution. The adult worms were designated as A. caucasica after careful identification based on the morphological and morphometric features, which was strengthened by previous descriptions by Schulz, (1926), Ortlepp, (1926) and Brede and Burger, (1977) [8,9,21]. In addition to the morphological and morphometric features previously listed, we reported the width of the esophagus (0.268–0.287 mm) and that of the cuticle (0.70–0.122 mm), which may help in the future identification of A. caucasica. Morphologically, Abbreviata species are closely related to each other [22]. In 1945, Morgan described the utility of uterine morphology (number and mode of origin of the uteri in the female worm) in the taxonomic classification. He classified species from the genus Abbreviata (n = 27) into more than three classes with two (didelphys), four (tetra-delphys), or more than four (polydelphys) branches. Of those, three were associated with monkeys: A. caucasica (Linstow, 1902), A. poicilomeira (Sandground, 1936), and A. multipapillata (Kreis, 1940) [4]. Based on the uterine morphology, A. poicilomeira and A. multipapillata are listed in class 5–15 G (5–15 uteri with common trunk), and 9–13 H (9–13 uteri without common trunk), respectively. However, A. caucasica can be easily differentiated by the fact that it is in class 4-D (4 uteri with common trunk). The morphologic-based classification of Physalopteridae members (e.g., Skrjabinoptera, Abbreviata, and Physaloptera) exclude some morphometric measurements from the taxonomic characters such Pathogens 2020, 9, 517 12 of 22 as the length of the esophagus, vulva position, and egg dimensions. These features seem to variate in the same species and are used only in exceptional cases such as P. squamatae (Harwood 1932), S. chamaeleontis (Gedoelst 1916), and S. simplicidens (Ortlepp 1922) [23]. As expected, our data confirmed the variability of these parameters within the A. caucasica (Table1). This reduced the utility of some commonly used indexes (a, b, and c) in nematode taxonomy [24]. In addition to the important taxonomic characters highlighted by Fain and Vandepitte (1964) (e.g., morphological features of the anterior end posterior ands, the number of uterine branches), the two adult females measured in the present study exhibited morphometric features of body and egg size close to those of P. mordens (Lipper, 1908), a species synonymous with A. caucasica (Linstow, 1902), where the body size of the female is 41–100 1.8–2.8 mm with a small egg of 45–49 × 32–34 µm [7]. Eggs were also similar to those reported by Poinar et al., 1972, where the size is × 35–40 25–35 µm [5]. However, A. caucasica (Linstow, 1902) has been described as having a small body × size of 24.75–23.84 1.12–1.18 mm and larger eggs of 57–62 42–45 µm. In contrast, the measurements × × from the study of Fain and Vandepitte (1964) showed that the A. caucasica (syn. P. mordens) adult females had a big body size of 108–117 mm and larger eggs of 60–65 45–55 µm (Table1). Furthermore, × the same authors confirmed and described the inconsistency of some measurements within this species [7]. Traditional taxonomic keys are known to be inconclusive for the taxonomic classification of nematodes [25] and should be confirmed by molecular barcoding, which circumvents the limitations of classical morphology-based classification [26]. The question then arises of whether A. caucasica (Linstow, 1902) is the same specie as P. mordens (Lipper, 1908), as indicated by Ortlepp (1926) and Fain and Vandepitte (1964) using morphologic-based taxonomy [7,9]. To address this question, a molecular comparative characterization of the specimens from the studies of Schulz (1926) and Fain and Vandepitte (1964) [7,8] should be performed to confirm or refute the synonymy of these two species. In our study, we expanded the genetic data available for A. caucasica with sequences of mitochondrial and nuclear DNA (i.e., cox1, 12S rRNA, 16S rRNA, 18S rRNA, and ITS2 genes), though the genetic characterization was based on cox1 and 18S rRNA genes, due to the limited data on other gene sequences of Physalopterida members in the GenBank database. The molecular analyses carried out in this study such as the phylogenetic comparisons of cox1 and 18S rRNA genes, the TCS network analysis of the cox1 gene, and the Bayesian inference of both cox1 and COI sequences confirmed that A. caucasica is monophyletic with Physaloptera species (Figures4A,B and5). cox1 and 18S rRNA genes are widely used as markers for the molecular barcoding of nematodes [27] with cox1 sequences of relevance in resolving taxonomic relationships among nematode species [27,28]. This gene is described by an interspecific nucleotide pairwise distance (INPD) of 16% to 27.8% between nematodes species [29]. The description of new species from the genus Physaloptera as well as the recording of new hosts has quickly evolved over the last decade [30–37]. However, there is a lack of additional data on the epidemiology, life cycle, clinical signs, and description of larval stages in intermediate hosts, which impedes progress in the understanding of these parasites. This is also related to the limited diagnostic and monitoring methods, which has for long time been exclusively based on the identification of eggs in feces [1]. Abbreviata caucasica appears to be capable of living attached to the wall of the digestive tract between the esophagus and the small intestine in human and non-human primates [1,33]. However, clinical features of A. caucasica infestation in chimpanzees remain unknown at this time and further studies are needed to identify such features [6]. We developed a specific 12S qPCR-based assay for the detection of A. caucasica from biological samples and potential intermediate hosts, though the unique Abbreviata species DNA and target sequence from A. caucasica used to confirm the assay specificity may represent a limitation of the assay. In contrast, the newly cox1 A. caucasica specific PCR could be used to assess the identification of A. caucasica from hosts exposed to a wide range of nematode infestations. Since the PCR replicated the same result as the qPCR (p = 0.25), both tools were highly sensitive and specific in detecting A. caucasica, Pathogens 2020, 9, 517 13 of 22 even the presence of coinfestations, avoiding the hard diagnosis based on egg identification. These tools can resolve problems related to the detection of larval stages from the intermediate and paratenic hosts and therefore avoid the sequencing identification by nematode generic primers. A detection limit as low as 1.13 10 3 eggs per gram of positive feces, regardless of consistency, solves the problems × − associated with conventional protocols requiring fresh equipment [38]. Data generated by qPCR showed a rate ranging from 0.2 to 1.4 eggs/g according to the fecal consistency, the best record being 113 egg/g. Appleton and Henzi (1993), reported the same results from baboons in Natal, South Africa, where egg output of A. caucasica ranged from 0.32 to 1.48 eggs/g with 215 eggs/g as the best record [39]. These observations highlight the usefulness of the qPCR quantification protocol we developed to evaluate the load of A. caucasica eggs. We therefore developed a 5S pan-Nematoda qPCR for the global exploration of nematode infestations from different biological samples. The absence of A. caucasica DNA from all environmental samples could be explained by the fact that they were not contaminated by the feces of infested hosts. However, despite the absence of A. caucasica DNA in the termite (Isoptera spp.) specimens that we tested, we cannot be sure if they are involved in the life cycle of A. caucasica or not. Termites (Isoptera) are the intermediate host of several nematodes such as A. antarctica, achanthocephalans (Thorny-headed worms), and Heterakis gallinarum [40–42]. Poinar and Quentin, (1972) experimentally demonstrated the ability of Blatella germanica and Schistocerca gregaria to develop the infective stage of A. caucasica. However, the life cycle of this nematode remains largely unknown. More than 28 paratenic and second intermediate hosts are also suspected [6]. However, we cannot be sure whether the environmental samples from species included in the diet of the chimpanzee population in our study, screened here, are not implemented in the life cycle of A. caucasica even in the absence of its DNA from all specimens. Termites are known to be the most prevalent arthropod in the chimpanzee diet [43].

4. Materials and Methods

4.1. Study Site and Study Subjects Samples were collected at the Dindefelo Community Natural Reserve, located in the Kedougou region, southeastern Senegal, about 35 km from the town of Kedougou. The vegetation of the reserve is a sudano-guinean savanna woodland [44], one of the driest and more open habitats occupied by the species [45]. All chimpanzees live in multi-female/multi-male communities composed of flexible groups that fission and fuse [46]. At the time of data collection, some individuals were semi-habituated to observers, but the rest remained unhabituated and thus the exact community composition and size were unknown. Although the total home range of Dindefelo chimpanzees was not known, conspecifics living in savanna woodland habitats have extremely large home ranges (e.g., >85 km2,[47]). Based on size, the fecal samples analyzed in this study were assumed to belong to adults.

4.2. Fecal, Worms, and Environmental Samples Two expeditions to the Dindefelo Community Natural Reserve in Senegal were undertaken in order to collect the samples. During the first trip (August 2016), 49 fecal samples of the western chimpanzee (Pan troglodytes verus) (Figure7A) were collected from three localities in the reserve: Locality 1, three decomposing “degraded” fecal samples (12.382539, 12.287977); Locality 2, six − degraded fecal samples (12.381437, 12.290776); and Locality 3, thirty-eight fresh fecal samples − (12.379919, 12.296830). The fecal samples were collected and stored at 80 C. Two adult worms were − − ◦ recovered from two fresh feces in the field and stored in 70% ethanol. In shape and general appearance, these worms resembling Ascaris to the naked eye (Figure7B). All samples were transported to our laboratory at the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection for further examination, and the adult worms were sent to the parasitology laboratory of the Department of Veterinary Medicine (University of Bari, Italy), where they were subjected to morphological identification. During the Pathogens 2020, 9, 517 14 of 22 second expedition (August 2019), we targeted the potential contamination of these parasitic nematodes for the chimpanzees (e.g., the possible intermediate hosts that chimpanzees could eat or their water Pathogenssources). 2020 A, total9, x FOR of 113PEER environmental REVIEW samples including the main species from the chimpanzee’s14 of diet 21 were collected in the vicinity of chimpanzee sleeping sites and other areas frequented by the apes. andThese other included areas 47frequented termites, 42by soil the samples apes. These from included termite mounds, 47 termites, 21 plant 42 soil species, samples one samplefrom termite from a mounds,water source 21 plant used species, by the chimpanzees,one sample from a centipede, a water source and one used maggot. by the chimpanzees, All samples were a centipede, preserved and in one70% maggot. ethanol All and samples were transported were pres toerved our in laboratory 70% ethanol for analysis. and were transported to our laboratory for analysis.

Figure 7. Adult of A. caucasica nematode found in the fecal matter of wild chimpanzee from the FigureDindefelo 7. Adult Community of A. caucasica Natural Reserve, nematode Senegal. found (A in) Ath chimpanzeee fecal matter (Pan of troglodytes wild chimpanzee verus) in itsfrom natural the Dindefelohabitat. (B )Community Adult female Natura of A. caucasical Reserve,looks Senegal. like Ascaris (A) Awith chimpanzee smooth and (Pan elastic troglodytes body, strangled verus) in head, its naturalcircular habitat. mouth (blue(B) Adult arrow), female and incurved of A. caucasica tail (yellow looks arrow). like Ascaris with smooth and elastic body, strangled head, circular mouth (blue arrow), and incurved tail (yellow arrow). 4.3. Morphological Analysis of A. caucasica Adult Worms 4.3. MorphologicalThe female wormsAnalysis were of A. processedcaucasica Adult for morphometricWorms analysis. The body of the nematodes wereThe measured female worms and then were cut processed into three for pieces. morphometr The centralic analysis. part was Thesubjected body of the to nematodes DNA extraction were measuredfor molecular and identification.then cut into Thethree cephalic pieces. andThe caudalcentral ends part ofwas the subjected worms were to DNA fixed andextraction stained for in molecularlactophenol identification. solution to observe The cephalic anatomical and structures.caudal ends Digital of the images worms and were measurements fixed and werestained made in lactophenolwith an optic solution microscope to observe Leica® anatomicalDM LB2 with struct diures.fferential Digital interference images and contrast. measurements The software were Leicamade® withLASAF an 4.1optic was microscope used for the Leica image® DM analysis LB2 with process differential including interference the measuring contrast. of nematodes, The software which Leica are® LASAFprovided 4.1 in was micrometers. used for the The image identification analysis was process carried including out following the measuring the description of nematodes, made by Schulz,which are(1926), provided Ortlepp, in micrometers. (1926) and Brede The and identification Burger (1977) was [ 8carried,9,21]. out following the description made by Schulz,The (1926), observation Ortlepp, of (1926) structures and inBrede the cephalic and Burger region, (1977) the [8,9,21]. stout size of the nematode, a thick cuticle finelyThe striated, observation a large of cephalic structures collarette, in the cephalic the total region, length, the and stout the distancesize of the from nematode, the anterior a thick end cuticle to the finelyvulva striated, all confirmed a large the cephalic identification collarette, of this the helminth total length, as A. and caucasica the distance. from the anterior end to the vulva all confirmed the identification of this helminth as A. caucasica. 4.4. Identification of A. caucasica Eggs from Positive Feces 4.4. IdentificationThe exploration of A. ofcaucasicaA. caucasica Eggseggs from wasPositive investigated Feces from two fecal samples from which the adult wormsThe were exploration collected. of AA. formol-ethercaucasica eggs sedimentation was investigated method from of two fecal fecal concentration samples from was which used [the48]. adultEgg identification worms were wascollected. carried A outformol-ether according sedime to thentation key of Fainmethod and of Vandepitte fecal concentration (1964) [7], was while used the [48].differential Egg identification diagnosis was was performed carried out as according described to elsewhere the key of [49 Fain]. and Vandepitte (1964) [7], while the differential diagnosis was performed as described elsewhere [49].

4.5. DNA Extraction Genomic DNA was extracted from 200 mg of fecal samples, adult worms of A. caucasica, and environmental specimens using the QIAGEN DNA tissue kit (QIAGEN, Hilden, Germany) following the manufacturer’s recommendations. The extraction was performed after two lysis steps: (i) mechanical lyses performed on a FastPrep-24™ 5G homogenizer using high speed stirring for 40 s in the presence of the powder glass, and (ii) enzymatic digestion using the proteinase K in the

Pathogens 2020, 9, 517 15 of 22

4.6.1. Development of PCR Primer Sets The primer sets used in this study are listed in Table4. First, sequences of the cytochrome c oxidase I (cox1), 16S rRNA, 12S rRNA, and the internal transcribed spacer 2 (ITS2) genes were used to design primer sets targeting nematodes. For each PCR system, a fasta ﬁle was constructed from nematode sequences retrieved from the GenBank database. Sequences were aligned using BioEdit v7.0.5.3 software [50]. The highly conserved areas were submitted in Primer3 software v. 0.4.0 [51]. PCRs standardization was performed as described elsewhere [52]. Primers designed are reported in Table4.

Table 4. Primer sets used for the molecular characterization of A. caucasica.

Target Melting Elongation Primer Name Sequences 5 -3 Size (bp) Speciﬁcity Ref. 0 0 Gene Tm Time Fwd-ITS-793 TCGATGAAGAACGCAGCTA ITS2 420–750 57 C 1’ Rwd-ITS-1495 AGTTTCTTTTCCTCCGCTTAGTT ◦ Fwd-12S-Nem-1 AAGTTTGATTTTGGTTTTGGTTG 12S 680 58 ◦C 1’ Rwd-12S-Nem-681 CCATTGACGGATGGTTTGTA Pan- This study Fwd-16S-Nem-488 GCAGCCTTAGCGTGATGG Nematoda 16S 430 58 C 1’ Rwd-16S-Nem-918 TAAACCGCTCTGTCTCACGA ◦ dg.Fwd.COI.Nem.257 TTGGKGGTTTTGGWAATTGG Cox 1 1069 52 C 1’30” dg.Rwd.COI.Nem.1325 CCAGCAAAATGCAWAGGAAAA ◦ Fwd.18S.631 TCGTCATTGCTGCGGTTAAA Pan- 18S 1127–1155 54 C 1’30” [53] Rwd.18S.1825 GGTTCAAGCCACTGCGATTAA ◦ Nematoda Fwd.Abbrev.COI.51f TGATCAGGGTTGGGAGCTT Cox 1 550 53 C 1’ A. caucasica This study Rwd.Abbrev.COI.601r AAAAAGAACAATTAAAATTACGATCC ◦

In addition, primers Fwd.18S.631 and Rwd.18S.1825r, recently designed to amplify a partial fragment of the 18S rRNA gene of nematodes, were also used in this study (Table4)[ 53]. These genes were chosen in order to compare the relatedness with Physaloptera and parasitic nematodes available in the GenBank database.

4.6.2. Polymerase Chain Reaction, Sequencing and Phylogenetic Analysis All PCR reactions were carried out in a total volume of 50 µL, consisting of 25 µL of AmpliTaq Gold master mix (Thermo Fisher Scientific), 18 µL of ultra-purified water DNAse-RNAse free, 1 µL of each primer, and 5 µL of genomic DNA. PCR reactions with all systems were run using the following protocol: incubation step at 95 ◦C for 15 min, 40 cycles of 1 min at 95 ◦C, 30 s for the annealing at a different melting temperature for each PCR assay, and elongation for 45 s to 1 min and 30 s (Table4) at 72 ◦C with a final extension for 5 min at 72 ◦C. PCR reactions were performed in a Peltier PTC-200 model thermal cycler (MJ Research Inc., Watertown, MA, USA). The amplicons obtained from each gene examined were purified using the filter plate Millipore NucleoFast 96 PCR kit following the manufacturer’s recommendations (Macherey Nagel, Düren, Germany). Purified DNAs were subjected to the second amplification using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Then, BigDye PCR products Pathogens 2020, 9, 517 16 of 22 were purified on the Sephadex G-50 Superfine gel filtration resin prior to sequencing on the ABI Prism 3130XL. First, all nucleotide sequences were assembled and edited by ChromasPro 2.0.0. The absence of co-amplification of nuclear mitochondrial genes (numts) was verified for the cox1 DNA sequences, wherein the alignment was performed with the complete sequence of cox1 DNA from the P. rara mitochondrion sequence (MH931178) using the ClustalW application within Bioedit v.7.2.5. [50]. In addition, the visual verification of sequence chromatograms ambiguities, indels and stop codons of the translated sequences was performed using Chromas Pro 2.0.0 software as recommended [54]. Sequences amplified from the cox1, 16S, 12S, 18S rRNA, and ITS2 genes were subjected separately to a preliminary analysis using the Basic Local Alignment Search Tool (BLAST) [55]. The closely related sequences of Physaloptera and nematode species retrieved from the GenBank database were included in the study and a fasta file was constructed for each gene and then subjected to the alignment. In addition, alignment of nematode COI protein sequences was also performed. All alignments were conducted using the ClustalW application within Bioedit v.7.2.5. [50]. The conservation of amino acids between the COI sequences of A. caucasica relative to the sequences of Physaloptera was visualized on CLC Sequence Viewer 7 (CLC Bio Qiagen, Aarhus, Denmark). From the alignment of each gene examined, the interspecific nucleotide pairwise distance (INPD) was evaluated to estimate the genetic divergence between all species included. Furthermore, the interspecific amino acid pairwise distance (IaaPD) was reproduced for COI-protein sequence alignment. standard errors were obtained by a bootstrap procedure with 1000 replicates using the maximum composite likelihood model [56] and Poisson correction model [57] for nucleotide and protein sequence alignments, respectively. Evolutionary analyses were inferred on MEGA6 software [58]. DNA sequences of Necator americanus (AJ920348) and Ascaris sp. (KC839986) were chosen as out-groups for18S rRNA and cox1, respectively, according to the fast-minimum evolution tree on BLAST [55]. The corresponding COI protein sequence of Ascaris sp. (AGN72537) was maintained as an out-group for the COI-protein alignment. The best model parameters with the lowest score were selected to generate phylogenetic trees of aligned 18S and cox1 sequences as well as COI protein sequences by running the MrBayes algorithm on each model using Topaliv2.5 software [59]. The Bayesian phylogenetic tree [60] was inferred for nucleotide sequence alignments using the K80 (+G) [61] and GTR (+G, +I) [62] models, respectively, while the Bayesian phylogenetic tree was inferred on the COI protein sequence alignment using Mtmam (+G) [63]. All phylograms were generated with five runs for 1,100,000 generations, 25% of burn-in length, and 1000 for sample frequencies. In order to resolve the haplotype variations of Physaloptera species and A. caucasica, the Fasta file of the cox1 sequences was converted to the Nexus format using an online converter [64]. During the second time, a Templeton–Crandall–Sing (TCS) network phylogram [65] was inferred with a 95% connection limit and drawn with 1000 iterations using the PopArt software [66].

4.7. TaqMan qPCR for Nematoda Parasites Detection The 5S rRNA gene was selected for the development of a TaqMan qPCR as an exploratory tool targeting nematode parasites. This choice was based on its conservation among nematodes species [67] and its tandem repetition of 110 times, which improves the assay sensitivity [68]. The partial sequences (XR002251414, JX489168, HM641830, M27961, JX117890, LS997562, U32120, AP018154, LK928622, X16226) representing nematode members from Spirurina and Rhabditida clades were aligned against the 5S gene of some plathelminth worms (Schistosoma mansoni: XR001974633, Spirometra erinaceieuropaei: LN313518, Hymenolepis microstoma: LR215994) and vertebrate hosts (human: AC275639, dog: XR003137316, chimpanzee: XR002941379, horse: XR002802613). Primers: qNem.5S.1f 50-ACCACGTTGAAAGCACGMC-30; qNem.5S.110r 50-TGTCTACAACACCTSGRATTCC-30; Eurogentec (Liège, Belgium), and a TaqMan probe qNem.5S.38p 6-FAM-50-AGTTAAGCAACGTTGGGCC-30-TAMRA; Applied BiosystemsTM, were chosen from the highly conserved region specific for nematodes. Pathogens 2020, 9, 517 17 of 22

4.8. Quantitative TaqMan Real-Time PCR (qPCR) for A. caucasica Detection Sequences of the 12S rRNA gene amplified from the adult worms of A. caucasica were aligned with the closely related sequences of nematodes available in the GenBank using Bioedit v.7.2.5. [50]. The specific regions for 12S rRNA of A. caucasica were submitted in Primer3 software v. 0.4.0 [51] in order to design the following primers Phy.12S.f.204:50-GAATTGGATTAGTACCCAAGTAAGTG-30/Phy.12S.r.305: 50-TGTTCCAAAAATCTTTCTAAGATCAG-30 (Eurogentec, Liège, Belgium) and TaqMan probe: Phy.12S.242p. 6VIC-GCGGGAGTAAAGTTAAGTTTAAACC-TAMRA) (Applied BiosystemsTM), allowing the amplification of a fragment of 101 bp. Both qPCRs were tested in silico within the DNA databases of metazoans (taxid: 33208), vertebrates (taxid: 7742), bacteria (taxid: 2), Canidae (taxid: 9608), Felidae (taxid: 9682), and humans (taxid: 9605). This experiment was performed for both combinations of forward-reward and probe-reverse of each qPCR using Primer-BLAST [69]. Subsequently, the specificity was also validated in vitro against the genomic DNA extracted from adult worms of A. caucasica and DNA database including several nematodes, arthropods, laboratory-maintained colonies, hemopathogens as well as human, monkey, donkey, horse, cattle, mouse, and dog as described elsewhere [52]. All qPCR reactions included 5 µL of DNA template, 10 µL of Master Mix Roche (Eurogentec), and 3 µL of ultra-purified water DNAse-RNAse free. Concentration of each primer, UDG, and each probe was 0.5 µL. The TaqMan reaction of both systems was run using the same cycling conditions. This included two hold steps at 50 ◦C and 95 ◦C for 2 and 15 min, respectively, followed by 40 cycles of two steps each (95 ◦C for 30 s and 60 ◦C for 30 s). The qPCR reaction was performed in a CFX96 Real-Time system (Bio-Rad Laboratories, Foster City, CA, USA) after activating the appropriate dye readers for each qPCR system. A protocol for the quantification of eggs has been established to assess the analytical sensitivity of qPCR in the detection of fecal infestation. A 10-fold serial dilution of DNA extracted from 200 mg of fecal matter containing 113 eggs (Figure2) per gram (i.e., 22.6 eggs /100 µL of eluted DNA and 1.13 eggs/5 µL of qPCR reaction). Standard curves and derived parameters (PCR efficiency, Slope, Y-intercept, and correlation coefficient) were generated using CFX Manager Software Version 3 [70]. The molecular approaches described above were used to screen the presence of A. caucasica and other nematodes in chimpanzee fecal and environmental samples collected in a chimpanzee dormitory.

4.9. Conventional PCR Specific for A. caucasica The use of universal pan-Nematoda primers does not allow for the identification of species-specific DNA sequences due to a non-specific amplification in co-infestations. A specific cox1-based PCR was developed in order to complete the identification of A. caucasica from fecal samples. The specific region for A. caucasica was analyzed for the design of the primers COI.51f and COI.601r, targeting 550 bp of the cox1 gene (Table1). A. caucasica cox1 partial sequences herein amplified by the pan-Nematoda primers from the adult worms were aligned with Heliconema longissimum (AN: GQ332423) and Gongylonema pulchrum (AN: AP017685), representative members of Physalopteroidea and Gongylonematidae, respectively.

4.10. Molecular Survey of A. caucasica and Nematode Infestations in a Chimpanzee Population and the Environmental Samples DNA from fecal samples of chimpanzee (n = 48) and environmental samples (n = 113) were screened for the DNA of A. caucasica and nematode using the 12S rRNA A. caucasica and the 5S rRNA pan-Nematoda qPCR assays, respectively. Positive samples for A. caucasica were also subjected to ampliﬁcation and sequencing using the cox1 A. caucasica-speciﬁc primers. Pathogens 2020, 9, 517 18 of 22

4.11. Statistical Analysis XLSTAT Addinsoft version 4.1 (XLSTAT 2019: Data Analysis and Statistical Solution for Microsoft Excel, Paris, France) was used for the statistical analysis. Results of qPCRs analysis were used to set a database using the Microsoft Excel® program (Microsoft Corp., Redmond, Washington, USA). The effect of localities and fecal consistency on the infestation rates were tested using the Khi2 test or exact Fisher test. One-way analysis of variance (ANOVA) was performed to compare the predicted eggs from fresh and degraded feces. Negative samples and those with a studentized residual higher than 2.9 were removed before discarding the ANOVA test. McNemar’s test was used to compare the detection accuracy of the qPCR and conventional PCR of A. caucasica from the chimpanzee samples. Significance level was considered at alpha 0.05 for all analyses. ≤ 5. Conclusions A. caucasica measurements indicated the inconsistencies of certain indexes such as index a, b, and c (Table1) within this nematode, while it remains distinguishable from other Physaloptera species by the morphological features of the anterior and posterior ends as well as the presence of four uteri with a common trunk. However, the phylogenetic analyses showed that A. caucasica are clustered together with other monophyletic species of the Physaloptera genus. In the absence of strong morphological and epidemiological data, the species of Abbreviata may be re-classified as Physaloptera and a revision of the genus is needed. We developed specific and reliable molecular tools for the detection and egg quantification of A. caucasica from fecal samples. The tests can ultimately help to identify possible intermediates as well as paratenic hosts involved in the life cycle of A. caucasica. We therefore investigated its prevalence in a chimpanzee population from Senegal. Further studies are needed to clarify the epidemiology, circulation, life cycle, and possible pathological effects of A. caucasica, and the role of paratenic hosts or arthropods as intermediate hosts.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-0817/9/7/517/s1. Figure S1. Abbreviata caucasica partial COI-protein sequences alignment against Physaloptera species. The sequences of A. caucasica (selected box) obtained from adult worms were aligned against Physaloptera sequences available in the GenBank database. Residues were matched as dots. Conserved areas are indicated in blue, while the intensity of mutations is indicated by a foreground color (red to black). Figure S2. Quantification protocol of the 12S A. caucasica-specific qPCR. (A) Determination of detection limits and efficiency (eggs/g of fecal matter). (B) Standard curves generated from a serial 10-fold dilution of DNA. Author Contributions: Conceptualization, O.M. and D.R.; Field study, O.M., D.R., B.D., G.D., and C.S.; Methodology, O.M. and Y.L.; Software, Y.L.; Validation, O.M., D.O., and D.R.; Molecular analysis, Y.L. and H.M.; Parasitological analysis, Y.L., M.S.L., and D.O.; Data curation, O.M. and Y.L.; Writing—original draft preparation, Y.L.; Writing—review and editing, B.D., D.R., D.O., O.M., R.A.H.-A., and A.B. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program “Investissements d’avenir”, reference ANR-10-IAHU-03, the Région Provence-Alpes-Côte d’Azur and European funding FEDER PRIMI. Acknowledgments: We thank the Direction des Parcs Nationaux and the Direction des Eaux et Forests Chasses et de la Conservation des Sols for permission to work in Senegal. We thank Paula Alvarez Varona, Daouda Diallo, Mamadou F. Diallo, and Mamadou Samba Silla for help in the field in the Dindefelo Community Natural Reserve, Senegal. Conflicts of Interest: The authors declare no conflict of interest.

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